Engineering microbial production of many commercially useful products has both advantages and disadvantage when compared to traditional chemical routes or isolation from organisms that naturally produce the desired compound. Many of the advantages associated with recombinant microbial production over traditional chemical synthesis include 1) the ability to synthesize the desired compounds at ambient temperature (decreased energy costs), 2) use of less expensive, readily available, typically renewable, and less toxic raw materials, 3) the production of less environmental waste, 4) the ability to harness regioselective and stereoseletive chemistry frequently observed when using biological catalysts, and 5) decreased purification costs from organisms that naturally produce the desired compound, often in commercially insignificant amounts. However, recombinant microbial production of a desirable compound has some disadvantages as well, such as inadequate compound production, poor growth characteristics, inadequate precursor supply, catalyst robustness and stability, regulatory issues (use of antibiotic markers), and host cell toxicity issues observed as a result of genetic modification. One particular problem associated with producing hydrophobic/lipophilic compounds in many microorganisms is limited internal storage capacity. Many hydrophobic/lipophilic compounds tend to accumulate in internal hydrophobic compartments within the cell (for example, intracellular membranes). Accumulation of these materials within the various compartments tends to be limited, as excess accumulation may have adverse effects on the viability of the host organisms (i.e. disrupting normal membrane function, decreased growth, increased toxicity to host cell, etc.).
One method used to increase the storage capacity in recombinant host cells is to increase one or more storage components of the cell. Arechaga et al. (FEBS Lett. 482:215-219 (2000); WO 01/29236 A1) describes a method to alter the intracytoplasmic membrane content and composition by expressing the b and/or c subunit of ATP synthase. Membrane proliferation was induced, allowing elevated expression of genes encoding membrane proteins. Arechaga et al. do not describe a method to increase the intracellular storage capacity of hydrophobic compounds produced in microorganisms, especially non-proteinaceous compounds.
The intracellular storage of hydrophobic compounds (such as oils) is known to naturally occur in some organisms. For example, many plants store triacylglycerides (TAG) in oil bodies. These oil bodies consist of a phospholipid monolayer stabilized primarily with a unique plant protein called oleosin, along with other minor proteins surrounding the TAG core. Oleosins have a thumbtack-like architecture, with the “shaft” portion consisting of hydrophobic amino acids and the head exhibiting an amphipathic structure (in Biochemistry and Molecular Biology of Plants, Buchanan, B., Gruissem, W. and Jones, R., eds., American Society of Plant Physiologists, Rockville, Md., 2000, pp 17-18). Oleosins are required for significant accumulation of TAG in the oil bodies. The recombinant expression of plant oleosins in microorganisms for storage of hydrophobic/lipophilic compounds has not been reported.
Many commercially valuable hydrophobic/lipophilic compounds are naturally produced in microorgansisms. Additionally, microorganisms can be genetically engineered to produce the desired molecules. Examples of these hydrophobic molecules include, but are not limited to, hydrophobic peptides and compounds derived from isoprene such as carotenoids, quinones, dolichols, tocopherols, fatty acids (i.e. omega-3 fatty acids), terpenes, steroids, chlorophylles, polyhydroxyalkanoates, and natural rubber. Based on their hydrophobic/lipophilic nature, many of these compounds accumulate or associate near or within the hydrophobic portions of cellular membranes, leading to changes in membrane structure which may result in the loss of cell viability (Sikkema et al., Microbiol Rev., 59(2):201-222 (1995)). Accumulation of hydrophobic/lipophilic compounds, especially in recombinant microorganisms engineered to produce them at elevated levels, is frequently limited by the amount of internal storage capacity within the microorganism.
Carotenoids represent a class of hydrophobic compounds currently being produced in recombinant microorganisms. The genetics of carotenoid production are well-known and have been exploited to produce a variety of carotenoids in recombinant bacteria (Lee et al., Chem Biol, 10:453-462 (2003)). Various genetic modifications to the isoprenoid/carotenoid biosynthesis pathway have been employed to engineer bacteria, such as E. coli, to produce high levels of various carotenoids.
Carotenoids, such as β-carotene and astaxanthin, associate and/or aggregate with phospholipid monolayers and bilayers (Shibata et al., Chem Phys Lipids, 113:11-22 (2001)). As a result, the capacity to store carotenoids may ultimately be limited to the amount of available hydrophobic storage capacity. For example, it has been reported that one of the primary limitations associated with microbial carotenoid production, especially in bacteria such as E. coli, is the inability to accumulate commercially-useful levels of carotenoids, as is the case in many industrially suitable production hosts (Schmidt-Dannert, C. and Lee, P., Appl Microbiol Biotechnol, 60:1-11 (2002)).
It has been speculated that the limits for carotenoid production in a non-carotenogenic host, such as E. coli, had been reached at the level of around 1.5 mg/g dry cell weight due to overload of membranes and blocking of membrane functionality (although U.S. Ser. No. 10/735442 has recently reported levels up to about 6 mg/g dry cell weight). It has been suggested that the future focus of engineering E. coli for high levels of carotenoid production should be on formation of additional membranes and on genetic manipulations leading to novel carotenoid sequestering systems (Albrecht et al., Biotechnol Lett, 21:791-795 (1999)).
The problem to be solved therefore is to provide a method to increase the hydrophobic/lipophilic compound titer in a microbial cell, especially in recombinant microorganisms engineered to produce such compounds.